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- All Subjects: Molecular Biology
Description
Microbial electrochemical cells (MXCs) offer an alternative to methane production in anaerobic water treatment and the recapture of energy in waste waters. MXCs use anode respiring bacteria (ARB) to oxidize organic compounds and generate electrical current. In both anaerobic digestion and MXCs, an anaerobic food web connects the metabolisms of different microorganisms, using hydrolysis, fermentation and either methanogenesis or anode respiration to break down organic compounds, convert them to acetate and hydrogen, and then convert those intermediates into either methane or current. In this dissertation, understanding and managing the interactions among fermenters, methanogens, and ARB were critical to making developments in MXCs. Deep sequencing technologies were used in order to identify key community members, understand their role in the community, and identify selective pressures that drove the structure of microbial communities. This work goes from developing ARB communities by finding and using the best partners to managing ARB communities with undesirable partners. First, the foundation of MXCs, namely the ARB they rely on, was expanded by identifying novel ARB, the genus Geoalkalibacter, and demonstrating the presence of ARB in 7 out of 13 different environmental samples. Second, a new microbial community which converted butyrate to electricity at ~70% Coulombic efficiency was assembled and demonstrated that mixed communities can be used to assemble efficient ARB communities. Third, varying the concentrations of sugars and ethanol fed to methanogenic communities showed how increasing ED concentration drove decreases in methane production and increases in both fatty acids and the propionate producing genera Bacteroides and Clostridium. Finally, methanogenic batch cultures, fed glucose and sucrose, and exposed to 0.15 – 6 g N-NH4+ L-1 showed that increased NH4+ inhibited methane production, drove fatty acid and lactate production, and enriched Lactobacillales (up to 40% abundance) above 4 g N-NH4+ L-1. Further, 4 g N-NH4+ L-1 improved Coulombic efficiencies in MXCs fed with glucose and sucrose, and showed that MXC communities, especially the biofilm, are more resilient to high NH4+ than comparable methanogenic communities. These developments offer new opportunities for MXC applications, guidance for efficient operation of MXCs, and insights into fermentative microbial communities.
ContributorsMiceli, Joseph (Author) / Torres, César I (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Rittmann, Bruce (Committee member) / Arizona State University (Publisher)
Created2015
Description
The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis.
ContributorsDatta, Īpsitā (Author) / Vermaas, Willem Fj (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2015
Improving cyanobacterial hydrogen production through bioprospecting of natural microbial communities
Description
Some cyanobacteria can generate hydrogen (H2) under certain physiological conditions and are considered potential agents for biohydrogen production. However, they also present low amounts of H2 production, a reaction reversal towards H2 consumption, and O2 sensitivity. Most attempts to improve H2 production have involved genetic or metabolic engineering approaches. I used a bio-prospecting approach instead to find novel strains that are naturally more apt for biohydrogen production. A set of 36, phylogenetically diverse strains isolated from terrestrial, freshwater and marine environments were probed for their potential to produce H2 from excess reductant. Two distinct patterns in H2 production were detected. Strains displaying Pattern 1, as previously known from Synechocystis sp. PCC 6803, produced H2 only temporarily, reverting to H2 consumption within a short time and after reaching only moderately high H2 concentrations. By contrast, Pattern 2 cyanobacteria, in the genera Lyngbya and Microcoleus, displayed high production rates, did not reverse the direction of the reaction and reached much higher steady-state H2 concentrations. L. aestuarii BL J, an isolate from marine intertidal mats, had the fastest production rates and reached the highest steady-state concentrations, 15-fold higher than that observed in Synechocystis sp. PCC 6803. Because all Pattern 2 strains originated in intertidal microbial mats that become anoxic in dark, it was hypothesized that their strong hydrogenogenic capacity may have evolved to aid in fermentation of the photosynthate. When forced to ferment, these cyanobacteria display similarly desirable characteristics of physiological H2 production. Again, L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation, which proceeded via a mixed-acid pathway to yield acetate, ethanol, lactate, H2, CO2 and pyruvate. The genome of L. aestuarii BL J was sequenced and bioinformatically compared to other cyanobacterial genomes to ascertain any potential genetic or structural basis for powerful H2 production. The association hcp exclusively in Pattern 2 strains suggests its possible role in increased H2 production. This study demonstrates the value of bioprospecting approaches to biotechnology, pointing to the strain L. aestuarii BL J as a source of useful genetic information or as a potential platform for biohydrogen production.
ContributorsKothari, Ankita (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Vermaas, Willem F J (Committee member) / Rittmann, Bruce (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2013